Recent evidence suggests that lipids in biological membranes do not mix uniformly, but instead form microdomains or rafts of different lipid composition. Domains that are enriched in cholesterol and sphingolipids can be isolated from membranes by the fact that they are insoluble in detergents such as Triton X-100. These detergent-resistant membranes (DRMs) are in the liquid-ordered phase, a state in which lipid acyl chains are ordered, extended, and tightly packed. Lipids and proteins that prefer an ordered environment are enriched in DRMs and probably also in liquid-ordered phase rafts in intact cells. Rafts have recently been shown to play important roles in signal transduction, especially in hematopoietic cells such as T cells and basophils. In both cases, signaling in response to antigen binding requires the presence of rafts. Thus, a better understanding of raft structure has important implications for immune diseases and the allergic response. In this proposal, we will address three questions of raft structure. First, how does clustering of proteins and lipids expected to be in rafts affect raft structure and the interaction between raft components? Second, how do transmembrane proteins associate with rafts? Because of the tight packing of lipids in rafts membrane-spanning alpha helical protein segments would not be expected to fit well into this ordered environment. However, a few transmembrane proteins are specifically targeted to rafts. In some cases, acylation is required for targeting of these proteins to rafts. Other raft-associated transmembrane proteins are not acylated, and the mechanism of raft association is not known. We will examine members of each class. The third broad question is the role of lipid composition in raft formation. We will address three subquestions; how rafts form in the sphingolipid-poor inner leaflet of the plasma membrane, raft size, and the role of sterol structure in promoting raft formation. We will address all of these questions using several assays for raft structure, and for the affinity of individual components for them. We use detergent-insolubility and the fluorescence quenching assay that we have developed previously, and will work to develop additional detergent-free assays of raft structure. With these tools, we should obtain a better understanding of these important membrane specializations, and how the association of proteins with them is important in health and disease.